Although our immune system is capable of discriminating healthy cells from tumor cells and infectious agents, it sometimes fails in appropriately recognising and reacting to the problem. Therefore, medical science has focused on the development of several strategies that aid the immune system in the surveillance and elimination of tumor cells and infectious agents. Dendritic cells (DCs) are antigen-presenting cells (APCs) which are known as key players in the instigation of immune responses and much effort has been put in the exploitation of DCs in immunotherapy. In the case of cancer for example, the aim is the induction and perpetuation of a tumor specific immune response by eliciting effector T cells that can specifically decrease tumor load and induce immunological memory to control tumor relapse. Once targetable tumor associated antigens (TAA) have been identified, they can be used to load the professional APCs, i.e. the dendritic cells, either in vivo or ex vivo.
Different antigen formats have been assessed with regards to DC for in vivo or ex vivo immunotherapy such as peptides, proteins, whole tumor cell extracts, plasmid DNA or mRNA. Among these approaches, antigen-encoding mRNA is emerging as particularly promising. The advantage over the classical vaccination with peptides is that mRNA encodes the genetic information for the whole antigen. The full-length antigen is processed and all available epitopes are presented in the MHC molecules of the patient, without the need to determine HLA specific peptides. No patients need to be excluded from the treatment because the available peptides do not match their HLA type. In addition, mRNA does not pose the risk of genomic integration giving it a favourable safety profile compared to DNA or viral vectors. Due to its transient nature, mRNA is only expressed during a short period of time and is eventually degraded into natural products. Furthermore, mRNA acts as its own adjuvant, prompting co-stimulatory signals, which is advantageous in the context of mRNA-based immunotherapy. Two routes for exogenous mRNA delivery into DCs have been applied: either ex vivo with subsequent adoptive transfer of transfected DCs or by direct administration of mRNA and uptake in vivo.
A study performed by Diken et al. (2011) highlights that the maturation stimulus and/or timing of its delivery have to be selected carefully as the uptake of mRNA is dependent on macropinocytosis, a function of immature DCs that is lost upon DC maturation. Consequently, co-delivery of classical maturation stimuli, such as lipopolysaccharide (LPS), with TAA mRNA has a negative impact on the bioavailability of the antigen, a parameter that co-determines the induction of antigen-specific T cell responses (Van Lint 2012; Diken 2011). To date two different strategies have been explored to simultaneously load the DCs with TAA mRNA and activate them in vivo.
Fotin-Mleczek et al. (2011) described a two-component system containing free- and protamin-complexed mRNA, providing an antigen source for adaptive immunity together with enhanced triggering of the pathogen recognition receptor, TLR7. This immunization strategy resulted in the induction of a strong anti-tumor immune response and in sustained memory responses, which is important, as memory T cells should avoid tumor re-appearance.
Bonehill et al., 2008 evaluated the use of specific combinations of mRNA for adjuvant purposes, initially for the activation of ex vivo generated DCs but equally applicable for direct administration and uptake in vivo (Bonehill, 2008). This has lead to a patent application (WO2009034172) in which the inventors describe that the T cell stimulatory capacity of antigenic-peptide pulsed antigen presenting cells or antigen presenting cells (co-) electroporated with an mRNA encoding a TAA can be greatly enhanced by providing them with different molecular adjuvants through electroporation with a mixture of mRNA or DNA molecules encoding two or more immunostimulatory factors. Proof of concept is provided that such modified antigen presenting cells pulsed with a target-specific peptide or co-electroporated with mRNA encoding a target-specific antigen can stimulate antigen-specific T cells both in vitro and after vaccination and thus form a promising new approach for anti-tumor, anti-viral, anti-bacterial or anti-fungal immunotherapy. A preferred combination of immunostimulatory factors used in the invention is CD40L and caTLR4, or CD40L and CD70. In other preferred embodiments, the combination of CD40L, CD70 and caTLR4 immunostimulatory molecules is used, which is called “TriMix” hereinafter.
The present invention relates to an RNA transcription vector containing a 5′ translation enhancer sequence and a 3′ nuclear retention sequence. The vector according to the present invention, shows an unexpected improvement in expression of the proteins encoded by the in vitro transcribed mRNA in comparison with an empty pUC vector, or with vectors that contain either a translation enhancer or a nuclear retention sequence. These improvements are in particular due to the simultaneous presence of the two components: a translation enhancer and a RNA stabilizing sequence in the vector, and the incorporation thereof in the thus obtained expression product. Furthermore, in vivo application of TriMix mRNA obtained from the vector of the present invention in a mouse cancer model results in a slower growth of tumors and an increased life expectance of said mice.